*highlights (for review)
TRANSCRIPT
Highlights
Core-shell structured TiO2-SiO2 nanoparticles of varying shell thickness were synthesized as
photo-killing agents.
The effect of the silica shell thickness on the photoreactivity, cytotoxicity, haemocompatibility and
photo-killing ability of the TiO2 nanoparticles was investigated.
Strong photo-killing effect and enhanced cytocompatibility were achieved by controlling the silica shell thickness to 5.5 nm.
*Highlights (for review)
Graphical abstract:
TEM image of core-shell structured TiO2-SiO2 nanoparticles (TS1) (Left), showing enhanced
compatibility to L929 cells (Middle) and effective killing ability of the cells upon photoexcitement
(Right), in comparison with the control nanoparticles (P25).
P25: non-coated TiO2
TS1: silica-coated TiO2
P25
TS1
TS1
*Graphical Abstract (for review)
Controlling silica coating thickness on TiO2 nanoparticles for effective
photodynamic therapy
Xiaohui Fenga‡
, Shaokun Zhangb‡
, Xia Loua*
a Department of Chemical Engineering, Curtin University, WA 6102 Australia
b Department of Orthopedics, The First Hospital of Jilin University, China
‡ These authors contributed equally to this work.
*Corresponding author. Tel.: +61892661682; fax: +61892662681.
Email address: [email protected].
*Revised Manuscript
2
ABSTRACT
Photosensitive nanoparticles are useful in developing phototherapeutic agents for targeted
cancer therapy. In this paper, core-shell structured titanium dioxide-silica (TiO2-SiO2)
nanoparticles, with varying shell thickness, were synthesized. The influence of the silica shell
thickness on the photoreactivity, cytotoxicity and photo-killing ability of the TiO2
nanoparticles was investigated. Silica coating reduced the photocatalytic reactivity but
improved the cytocompatibility of the TiO2 nanoparticles. This effect was amplified with
increasing silica shell thickness. When the silica thickness was about 5.5 nm, the coated TiO2
not only retained a high level photodynamic reactivity, comparable to the non-coated TiO2
nanoparticles, but also demonstrated an improved cell compatibility and effective photo-
killing ability upon the mouse fibroblast cells (L929).
Keywords: Silica-coated TiO2 nanoparticles; Silica coating; TiO2 nanoparticles; Photo-killing
agent; Photodynamic therapy
3
1. Introduction
Nanoparticles of TiO2 have attracted increasing attention in life sciences since the first
report of photocatalytic disinfection by Matsunaga et al. in 1985 [1]. When being irradiated
by ultraviolet (UV), the TiO2 nanoparticles can be photoexcited to produce a negative
electron (
CBe ) in the conduction band and a positive hole (
VBh ) in the valence band. In an
aqueous environment, the photo-induced electrons and hole pairs react with oxygen or water
to generate reactive oxygen species (ROS) such as hydroxyl (HO∙) and superoxide radical
(∙O2-). These reactive species are powerfully oxidative and can destroy the structure of
various organic molecules, therefore, having found extensive applications in the removal of
infectious molecules and organic pollutants. TiO2 nanoparticles also have been regarded as a
potential photosensitizing agent for photodynamic therapy (PDT) [2-5]. The ROS generated
from the photoexcited TiO2 nanoparticles can react with cell membranes and cell interiors,
leading to toxic responses and/or death of cells [6]. Many investigations on
photodecomposition of tumour cells have been undertaken recently. Cai et al. reported that
TiO2 nanoparticles completely killed HeLa cells with UV irradiation [7]. Stefanous et al. also
indicated that photoexcited TiO2 nanoparticles efficiently inhibited the aggregation of
platelets, which led to discontinuation of haematogenous metastasis [8]. The photo-killing
effect of nitrogen-doped TiO2 nanoparticles in the visible region also has been reported [9].
The research mostly has been limited to lab investigations. The clinical application of TiO2
nanoparticles has been hampered by problems such as insufficient selectivity and low
efficiency resulting from the lack of cell-specific accumulation of TiO2 on cancer cells [10].
In addition, the metal ions exposed on the surfaces of nanoparticles can lead to metal toxicity
in cells [10, 11]. The insolubility of TiO2 in water is also a problem as the nanoparticles tend
4
to aggregate in a physiological environment, leading to a reduction of surface area and
reactivity of the particles [12, 13].
Coating nanoparticles with silica, using tetraethoxysilane in the presence of ammonia, has
been used widely to improve the dispersion and the cell compatibility of various
nanoparticles in a physiological environment [14-16]. The method is simple and can be
operated at ambient temperature. The resultant nanoparticles consist of a core made of the
base nanoparticle and a shell made of silica, therefore, known as a core-shell structure. Silica,
as a shell, can act as a protector to reduce the influence of the outer environment upon the
core nanoparticles. Since the silica surface is electrostatically stable, it also improves the
dispersion of the core nanoparticles [17]. Although the overall particle stability and
dispersibility can be improved after silica coating, the properties of the core component, such
as reactivity and thermal stability, also may be modified [18, 19]. This article presents our
investigations regarding the influence of the silica coating on the photoreactivity, the
cytotoxicity and the photo-killing ability of TiO2 nanoparticles. Silica-coated TiO2
nanoparticles were synthesised at room temperature via Stöber method using commercially
available TiO2 nanoparticles (Degussa P25) and tetraethoxysilane (TEOS) as reactants. The
silica shell thickness was finely tuned through alteration of the TiO2-to-TEOS ratio in the
reaction mixture. The obtained core-shell structured nanoparticles were examined using a
field emission scanning electron microscope (FESEM), a transmission electron microscope
(TEM) equipped with an energy dispersive spectroscope (EDS), and a Fourier transform
infrared (FTIR) spectroscope. The photoreactivity of the silica-coated and non-coated TiO2
nanoparticles was first examined through the photo-degradation of phenol, a compound that
can be degraded into carbon dioxide, water and corresponding mineral acids in the presence
of photocatalysts under UV light [20]. Cytotoxicity and haemocompatibility were also
assessed. Following these examinations, silica-coated TiO2 nanoparticles with an optimal
5
shell thickness, having retained good photoreactivity, were selected for the photo-killing
effect investigation. The results are being used in the development of a targeted
photodynamic nanosystem for cancer cells.
2. Experiment
2.1. Materials and chemicals
Degussa P25 (TiO2 nanoparticles consisting of 80% anatase and 20% rutile) was
purchased from Degussa. The average size of the nanoparticles was 25 nm. Tetraethoxysilane
(TEOS) (99.999%), ammonia aqueous solution (4.13%) and ethanol (99.5%) were purchased
from Sigma-Aldrich. All chemicals were reagent grade and used without further purification.
Deionized water was used in this investigation.
2.2. Synthesis of TiO2-SiO2 core-shell nanoparticles
To a mixture of deionized water (20 ml) and ethanol (60 ml), 0.5 g P25 and 1.0 ml
ammonia solution were added. The mixture was dispersed using ultrasonic vibration for
about 30 min. Twenty ml of ethanol, containing various concentrations of TEOS, was added,
dropwise, to the dispersion. This took about 30 min. The concentrations of TEOS added into
the dispersion were 8.97 mM, 17.93 mM, 26.90 mM and 36.0 mM. These concentrations
yielded theoretical molar ratios of Si/Ti of 0.14, 0.29, 0.43 and 0.57 in the products,
respectively. After stirring for 2 h, the reaction mixtures were centrifuged at 7500 rpm for 5
min. The liquid in the centrifuge tube was removed and the resultant silica-coated TiO2
6
nanoparticles were washed with ethanol (three times) to remove the excess amount of
reactants and then dried overnight at room temperature under vacuum. The silica-coated TiO2
nanoparticles were denoted as TSX (X=1, 2, 3 and 4). The reaction is shown in Scheme 1.
Scheme 1. The schematic representation of TiO2 to silica-coated TiO2 via the TEOS
hydrolysis and condensation reaction. The theoretical molar ratios of Si/Ti in TS1-4 were
0.14, 0.29, 0.43 and 0.57, respectively.
2.3. Characterization of TiO2-SiO2 core-shell nanoparticles
FTIR analysis was conducted on a Perkin-Elmer Spectrum 100 using the KBr pellet technique at room
temperature. The samples were mixed with dried KBr, using a mortar and pestle, and then the mixture
was pelletized under vacuum. All the spectra were recorded in the range of 4000-400 cm-1
at a
resolution of 4 cm-1
. FESEM (Zeiss Neon 40EsB FIB-SEM) was used to examine the morphology of
P25 and the obtained TiO2-SiO2 nanoparticles. The samples were dispersed in deionized water using
ultrasonic vibration and the solutions were deposited onto an aluminium stub where a platinum
coating (2 nm) was applied as a conducting material. FESEM images of the nanoparticles were
recorded at an accelerating voltage of 5 kV. The size of the nanoparticles was measured using
the in-built Zeiss operational software, SmartSEM, that is linked to the magnification bar of
the obtained images. The detailed morphology and chemical composition of the nanoparticles were
further examined using TEM (JEOL JSM 2011) equipped with an energy dispersive spectroscope
(EDS). Prior to the TEM examination, the nanoparticles were dispersed in ethanol using ultrasonic
vibration with an approximate concentration of 10 μg/ml. They were then distributed on carbon-
coated copper grid for examination.
7
2.4. Photoreactivity study
Examination of the photoreactivity of the produced core-shell nanoparticles was
conducted in a 1L double-jacket reactor, as shown in scheme 2. A water bath was connected
to the reactor through a pump to maintain the reaction temperature at 25± 0.5 ◦C and a
magnetic stirrer was used for mixing. The UV irradiation was facilitated by a MSR 575/2
metal halide lamp (575 W, Philips) with the wavelength in a range of 315 to 1050 nm. In
brief: a preferred amount of P25 and TSX (X=1, 2, 3, 4) was added to 200 ml of aqueous
phenol solution and stirred for 30 min in the dark to obtain a homogeneous solution. Then the
light was immediately switched on. At set time intervals, 3 ml of solution was withdrawn by
a syringe and filtered using a 0.45 μm Millipore film. The concentration of phenol in the
withdrawn samples was measured using a HPLC (Varian) with a UV-detector at the
wavelength of 270 nm. The column was C-18 and the mobile phase was 30% acetonitrile and
70% deionized water. The amount of P25 used for photocatalytic reaction was 0.2 g resulting
in a concentration of 1.0 g/L. To maintain the same concentration for TiO2, the added
amounts of TS1-4 were 0.22 g, 0.24 g, 0.26 g and 0.28 g, respectively.
8
UV
25 oC
Magnetic Stirrer
Reactor
Circulated Water
Scheme 2. Photocatalytic reaction set-up
2.5. Cell culture and nanoparticle preparation
Primary adherent mouse fibroblast connective tissue cells (L929) were purchased from
ATCC (USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM,
Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS, Sigma Aldrich). The
cells were maintained at 37 oC in a humidified incubator with 5% CO2 and 95% air.
Nanoparticles of P25, TS1 and TS4 were suspended in phosphate buffered saline (PBS), each
at concentrations of 12.5, 25, 50, 100 and 200 μg/ml, for further investigation.
2.6. Cytotoxicity study
The cell cytotoxicity was measured using the 3-(4, 5-dimethylthiazol-2-yl)-5-(3-
cayboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay [21]. Briefly, L929
cells were plated at a density of 1x104 cells per well in a 96-well plate in 200 µl of medium
and incubated at 37 oC in 5% CO2 atmosphere. After overnight incubation, the medium in the
wells was replaced with fresh medium, described previously, containing the nanoparticles at
9
concentrations of 12.5, 25, 50, 100 and 200 μg/ml. A control experiment was conducted
using cells treated with complete medium containing no nanoparticles. After 6 h and 24 h of
incubation at 37 oC under 5% CO2, the cell culture medium was removed and the plate was
washed with PBS three times. 200 µL of MTS reagent was subsequently added to each well
and the cells were incubated for 3 h under the same conditions. After the treatment, the
absorbance of formed formazan at 490 nm was measured by microplate reader. The cell
viability was calculated using the following equation [22]:
%100
)(490
490 controlOD
sampleODviabilityCell (1)
where OD490 (sample) is the optical density of cells treated with various concentrations of
nanoparticles and OD490 (control) is the optical density of cells incubated with medium only.
All the experiments were performed in triplicate in the dark. Cell morphology was
photographed at the two time intervals using an Olympus BX61 microscope. The
magnification was set at 5 for all samples.
2.7. Haemolysis assay
Fresh blood was obtained from a rabbit. Red blood cells (RBCs) were isolated from
plasma using a refrigerated centrifuge at 1500 rpm for 15 min at 4 oC. The RBCs were
further washed three times with sterile PBS by centrifugation until the supernatant was clear.
Then 100 µl of the obtained particle suspensions in PBS at concentrations of 12.5, 25, 50,100
and 200 µg/ml were added to 100µl of the RBCs suspension. The mixtures were incubated at
37 °C for 1 h under constant shaking, and subsequently centrifuged at 1500 rpm for 15 min.
10
After the treatment, 100 µl of supernatant from each centrifuge tube was used to analyse
haemoglobin release by microplate reader at the wavelength of 576 nm. Control experiments
were performed under the same experimental conditions. 100 µl of the RBCs suspension was
added to 100 µl of PBS as a negative control and to 100 µl of 0.5 % Triton X-100 as a
positive control. The percentage haemolysis was calculated using the following equation [23]:
%100(%)576576
576576
controlNegativeControlPositive
controlNegativeSample
ODOD
ODODHemolysis (2)
where OD576 sample, OD576 Negative control and OD576 Positive control are the optical densities of
haemoglobin released from RBCs treated with, respectively, nanoparticles, PBS buffer and
medium containing 0.5 % Triton X-100.
2.8. Photo-killing effect
The photo-killing effect of TiO2-SiO2 nanoparticles on L929 cells was examined as
follows. L929 cells, at the density of 1x104 cells per well, were seeded in a 96-well plate in
RPMI-1640 medium (Lonza) and incubated overnight, in the dark, with 5% CO2 and
humidified atmosphere. Then the medium was replaced with the medium containing P25 or
TS1 nanoparticles, each at concentrations of 12.5 and 25 μg/ml. A control experiment was
conducted using cells treated with the medium containing no particle. After 24 hours, the
cells were exposed to UV light (365nm, 50W) for 20 min. The cells were incubated for
another 24 h in the dark and then the viability of the cells was tested using the MTS assay as
described above. The cell viability was expressed as the ratio of the number of viable cells
remaining after UV irradiation to those present in the samples without UV irradiation.
11
2.9. Statistical analysis
The experimental data of the aforementioned cellular work were analysed utilizing the
student’s T-test and the results were presented as mean ± standard deviation. The standard
deviation values are expressed as error bars. Statistical significance was considered at a
probability of p < 0.05.
3. Results and Discussion
3.1. Characterization of P25 and TS1-4
Four core-shell structured TiO2-SiO2 nanoparticles, TS1, TS2, TS3 and TS4, were
synthesized via the Stöber method using varying ratios of P25 and tetraethoxysilane (TEOS).
FESEM images showed that the resultant nanoparticles were sphere-like and slightly bigger
than P25 (Fig. 1). The analysis of 30 particles from each of the FESEM images indicated that
the non-coated TiO2 (P25) had a mean diameter of 241 nm, which is in agreement with the
product specification provided by the supplier. The coated particles were about 352 nm,
391 nm, 422 nm and 462 nm for TS1, TS2, TS3 and TS4 respectively, indicating an
increased shell thickness of 5.5 nm, 7.5 nm, 9 nm and 11 nm in TS1-4. The gradual increase
in the particle size, and therefore the shell thickness, was due to the increase of TEOS
concentration (Si/Ti ratio) in the reaction mixtures.
The TEM micrographs in Fig. 2 (Left) further demonstrated the core-shell structure of the
TiO2-SiO2 nanoparticles. EDS analysis of TS1 indicated the presence of silica on the surface
12
of the TiO2 nanoparticles (Fig. 2 Right). Further analysis of these nanoparticles using FTIR,
shown in Fig. 3, confirmed the formation of the silica network through the strong adsorption
peaks at 1065 and 1180 cm-1
which correspond to the asymmetric Si-O-Si bending and
stretching vibration, respectively. The peak at 1625 cm-1
is attributed to O-H bending
vibration of the surface silanol group of the silica gel (-Si-O-H groups). There is also a weak
adsorption peak at 950 cm-1
, attributed to the flex vibrations of Si-O-Ti, indicating that the
TiO2 core is connected with the silica shell through a chemical bond [24]. The weak peaks at
2920 and 2853 cm−1
arise from the symmetric and asymmetric stretching vibrations of the
−CH2 and –CH3 groups, which indicate the presence of the intermediate reaction product
(OR)3Si(OH) (R= -CH2CH3) (Scheme 3) [25]. As shown in Fig. 3, these peaks are generally
stronger in the coated TiO2 containing the higher silica component. The strong signals at
approximately 650 and 500 cm-1
are due to the Ti-O stretching vibration, confirming the
presence of TiO2 in the nanoparticles [26, 27].
Scheme 3. The hydrolysis reaction of TEOS and the subsequent condensation of the
intermediate reaction product of (OR)3Si(OH) [28].
3.2. Photocatalytic reactivity of P25 and TS1-4
The photodegradation reaction of phenol was carried out in the presence of P25 and TS1-
4, using a concentration that was equivalent to 1.0 g/L P25. As shown in Fig. 4, 94.5% of
phenol was degraded after 60 min of irradiation when P25 was used. When TS1-4 were used,
13
the phenol degradation yields were reduced to 86.6, 58.7, 50.5 and 36.7%, respectively,
indicating a reduction in photocatalytic reactivity of the coated TiO2 nanoparticles. The
apparent degradation rate constant of these nanoparticles was estimated using the Langmuir-
Hinshelwood equation [29]:
tc
c
o
ln (3)
where k is apparent reaction rate constant in the unit of time-1
, C0 is the initial concentration
and C is the concentration at time t.
The computed k values for P25, TS1, TS2, TS3 and TS4 were 0.0498, 0.0351, 0.0152,
0.0113 and 0.0075 min-1
, indicating a rapid decrease in the photo-degradation rate with
increase in shell thickness (Fig. 4 insert). The reduced photo-catalytic reactivity can be
attributed to the shielding effect of the silica shell formed on the surface of TiO2
nanoparticles. That is to say that the silica shell acted as a barrier to prevent the migration of
reactive radicals to the surface of the nanoparticles, which led to a decrease of active radicals
2O and OH for the oxidation of phenol [18].
It should be noted that the calculation was carried using the experimental data up to 60
min. The least squared R values for each fitting were 0.95, 0.99, 0.99 and 0.96. When the
reaction was extended to 90 min, phenol degradation results for P25, TS1, TS2, TS3 and TS4
were 99.9, 97.6, 87.5, 78.7 and 58.9%, respectively. This indicates that, with a slight increase
in reaction time, TS1 (with a shell thickness of 5.5 nm), is able to yield a complete photo-
degradation of phenol, similar to P25. Based on these observations, TS1, TS4 and P25
nanoparticles were selected for the cytotoxicity, haemocompatibility and photo-killing
investigations.
14
3.3. Cytotoxicity and haemocompatibility of P25, TS1 and TS4
The MTS assay was employed to determine the viability of L929 cells treated with P25
(the non-coated TiO2 nanoparticles), TS1 and TS4 (the silica-coated TiO2 nanoparticles) of
varying concentrations (12.5, 25, 50, 100 and 200 μg/ml). The culture was carried out in dark
for various time intervals. Results are shown in Fig. 5. When the nanoparticle concentration
was below 200 μg/ml, the viability was generally the same for both untreated cells and cells
treated with P25, TS1 and TS4. Incubation for 6 hours or 24 hours did not make any
difference. When the concentration of nanoparticles was increased to 200 μg/ml, no
significant difference in cell viability was observed at 6 hours. However, when the incubation
time was extended to 24 hours, the P25-treated cell viability was reduced to 82.0% (relative
to control) (p<0.05), whilst the TS1- and TS4-treated cell viability was 96.5% and 100%,
suggesting a much lower cytotoxicity of the silica-coated nanoparticles. This can be
attributed to the silica coating being cytocompatible. The optical micrographs (Fig.6) of the
P25-treated cells, the TS1-treated cells and the TS4-treated cells at 24 h further confirm the
results obtained from the MTS assay. As shown in Fig. 6(b), there was a portion of red-
stained nuclei in the cell culture, indicating that the cells had undergone apoptosis after
exposure to P25 nanoparticles. However, under the same conditions, there were scarce red-
stained nuclei in both control cells (Fig. 6(a)) and those treated with TS1 and TS4
nanoparticles (Fig.6(c) and 6(d)). A similar result was reported by Mbeh et al. [16] in a
biocompatibility study of silica-coated magnetite nanoparticles. They have concluded that a
silica shell significantly reduced the cellular toxicity of pure magnetite nanoparticles.
In vitro haemolysis assays of P25 and TS1 were conducted at the same concentration
range. Triton X-100 was used to induce full haemoglobin release. As shown in Fig. 7, the
15
haemolysis percentage was well below 5.0% across the range of investigated concentrations.
This indicates that P25, TS1 and TS4 exhibit excellent haemocompatibility and can be further
used in vivo.
3.4. Photo-killing effect of P25 and TS1
In order to select an appropriate UV exposure time, the effect of UV irradiation on L929
cell viability was first tested. Fig. 8 shows that the surviving fraction of L929 cells generally
decreased with increasing irradiation time. When the irradiation time was over 40 min, the
surviving fraction of L929 cells was only 15.5%, suggesting UV irradiation is harmful to
L929 cells. At 20 min, the surviving fraction of L929 cells was 90.2%, showing a small
suppression in cell proliferation. Based on these results, the photo-killing effect of P25 and
TS1 nanoparticles were evaluated through the comparison of the viability of non-treated cells,
with that of cells treated with P25 or TS1, after UV irradiation for 20 min. As shown in Fig.
9, the viability of the L929 cells was 90.2% relative to that of cells without exposure to the
UV light. However, the viability of L929 cells was reduced to 58.6% and 66.3% in the
presence of 15 µg/ml of P25 and TS1, and further to 56.5% and 58.9% when the
concentration of the nanoparticles was increased to 25 µg/ml. In comparison to the untreated
cells, a statistically significant (p0.05) photo-killing effect of both P25 and TS1 on L929
cells was demonstrated. P25 was slightly more powerful in killing the L929 cells at 12.5
µg/ml. However the killing effect became almost the same (p˃0.05) when the nanoparticle
concentration was increased to 25 µg/ml. It should be noted that the relative concentration of
TiO2 in TS1 is 90% of the apparent concentration in this experiment. These results further
suggest that TS1 and P25 exhibit a similar photo-killing effect on L929 cells under UV
irradiation.
16
4. Conclusions
In summary, core-shell structured TiO2-SiO2 nanoparticles of varying shell thicknesses
were synthesized and confirmed through SEM and TEM/EDS examinations. FTIR analysis
indicated the formation of Ti-O-Si chemical bonds on the surface of TiO2 nanoparticles. The
presence of a silica shell has improved the compatibility of TiO2 nanoparticles with L929
cells. Both coated and non-coated TiO2 nanoparticles exhibited good haemocompatibility.
Reduced photocatalytic reactivity was evident after the TiO2 nanoparticles were coated
with silica. This was compensated for by improved cell compatibility. When the silica shell
thickness was controlled to a minimum (5.5 nm in this study), the photocatalytic reactivity of
coated TiO2 (TS1) was very close to that of non-coated TiO2 (P25). The well-maintained
photoreactivity of TiO2 in TS1 was further demonstrated in cellular work in which both P25
and TS1 were able to reduce the viability of L929 cells to below 60% after a 20 min UV
irradiation. The nanoparticles have been further modified with various ligands for targeted
photodynamic therapy.
Acknowledgements
The authors would like to thank Dr Lihong Liu for her assistance in cell culture work. The
technical support by the Centre for Materials Research, Curtin University in operating the
SEM and TEM is also acknowledged. The Curtin Electron Microscope Facility is partially
funded by the University, State and Commonwealth Governments.
17
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Figure Captions
Fig. 1. FESEM micrographs of P25 and TS1-4.
Fig. 2. TEM images of P25 and TS1 (Left), showing the core-shell structure of TS1. EDS of
TS1 (Right), showing the presence of elemental Ti, Si and O. Cu is from the copper grid and
C is from both TS1 and the carbon film coated on the copper grid.
Fig. 3. FTIR spectra for P25 and TS1-4.
Fig. 4. Phenol concentration change with time (Insert: degradation rate change with shell
thickness).
Fig. 5. Cell viability after treatment with P25, TS1 and TS4 for (a) 6 h and (b) 24 h.
*p0.05 as compared to control (untreated) cells.
**p0.05 as compared to the P25 treated cells. All data are expressed as the mean ± SD and n=3.
Fig. 6. Light micrographs of L929 cells treated with (a) no nanoparticles, (b) P25, (c) TS1,
and (d) TS4. Incubation time = 24 h. The nanoparticle concentration = 200 μg/ml.
Fig. 7. Haemolysis assays of P25, TS1 and TS4 nanoparticles.
All data are expressed as the mean ± SD and n=3.
Fig. 8. Effect of UV irradiation time on viability of L929 cells.
*p0.05 as compared to cells without UV irradiation.
All data are expressed as the mean ± SD and n=4.
Fig. 9. Effect of photo-excited TiO2 and TiO2-SiO2 nanoparticles on cell viability. Irradiation
time was 20 min.
*p0.05 as compared to control.
All data are expressed as the mean ± SD and n=4.
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